Tomas Morosinotto, Matteo Ballottari, Frank Klimmek, Stefan Jansson and Roberto Bassi
A 2
The role of individual Lhca subunits in the stability of higher plant Photosystem I-Light harvesting I supercomplex
Tomas Morosinotto1,2, Matteo Ballottari1, Frank Klimmek3, Stefan Jansson3 and Roberto Bassi1,2
1 Dipartimento Scientifico e Tecnologico, Università di Verona. Strada Le Grazie, 15- 37134 Verona, Italy
2 Université Aix-Marseille II, LGBP- Faculté des Sciences de Luminy - Département de Biologie - Case 901 - 163, Avenue de Luminy - 13288 Marseille, France
3 Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, S-901 87 Umeå, Sweden
Running title: Antenna binding cooperativity to higher plants Photosystem I
Abbreviations used are: α(β)-DM: n-dodecyl-α(β)-D-maltoside; Car, Carotenoid; Chl, chlorophyll;
Lhca (b), light harvesting complex of Photosystem I (II); PSI (II), Photosystem I (II); WT, wild type.
Keywords: Photosystem, LHCI, Lhca, antenna, cooperativity
Abstract
We report on the results obtained by a biochemical analysis of Photosystem I in plants depleted in individual Lhca complexes. Our analysis showed that the lack of one Lhca complex affects the stability of the whole antenna system, demonstrating the cooperative nature of its association to Photosystem I. Strong interactions were shown between Lhca polypeptides and between antenna and core subunits which are important for the stability of the whole supercomplex. These interactions are stronger between respectively Lhca1-4 and Lhca2-3, consistently with their purification as dimers. Gap and linker pigments are proposed to play a fundamental role in mediating the interactions between antenna polypeptides. Differences between in the association of antenna complexes in Photosystem I and II are discussed. Lhca5, a minor component of the antenna system plays a role in the stability of the supercomplex.
Introduction
Photosystem I in higher plants mediates the light driven electron transport from plastocyanin to Ferredoxin. It is composed by two moieties: (i) a core complex, binding chromophores involved in charge separation and electron transport; (ii) an antenna moiety responsible for increasing the absorption cross-section. In higher plants, the core complex is composed by 14 subunits [1; 2] and it also binds about 100 Chl a and 22 β-carotene molecules [3; 4].
The antenna moiety is called LHCI: in higher plants it is composed by 4 major polypeptides encoded by nuclear genes Lhca1-4 [4; 5]. Two other Lhca gene sequences, Lhca5 and Lhca6, have been identified in Arabidopsis thaliana, as well as in other plant species [5]. Lhca5 gene product has been recently detected in thylakoids where it appears to be part of PSI-LHCI supercomplex although in sub-stoichiometric amounts [6; 7]. Lhca6 is highly homologous to Lhca2 and could be a pseudo-gene, but it might be expressed with a physiological role only in particular growth conditions (Klimmek, unpublished)
Recently, the structure of PSI-LHCI complex from Pisum sativum has been resolved by X-ray crystallography, showing the presence of one copy each of the Lhca1-4 polypeptide bound to one side of PSI core [4]. A unique feature revealed by this structure is the presence of chlorophyll molecules bound at the interface between LHCI subunits and between LHCI and the PSI core complex, defined respectively as “gap” and “linker” chlorophylls [4]. Biochemical fractionation of LHCI and PSI core showed that, besides Chl a, Chl b and carotenoid molecules are also found in this interfacial pigment pool [8].
In this work, we address the question of how LHCI binds to PSI core by performing a biochemical characterisation of the PSI-LHCI complex from Arabidopsis thaliana plants lacking each individual Lhca polypeptides, obtained by either insertion mutagenesis or RNA interference. Our analysis shows that the association of Lhca subunits to PSI core is strongly cooperative: in fact, when one subunit is missing the whole LHCI system is de-stabilised. On this basis, protein - protein interactions, mediated by gap and linker chlorophylls, are suggested to play an important role in LHCI binding to PSI core. These interactions appears to be particularly strong between Lhca1 - Lhca4 and Lhca2 - Lhca3, consistently with their isolation as dimers [9]. The subunit association in Arabidopsis PSI-LHCI complex is compared both with Photosystem II and with PSI from Chlamydomonas reinhardtii.
Materials and methods
Plant material and thylakoids purification
∆a1-a5 plants and growing conditions have been previously described in [6; 10]. Thylakoids have been purified from dark adapted plants as in [11].
Non denaturing gels (Deriphat PAGE).
Non-denaturing Deriphat-PAGE was performed following the method developed by Peter and Thornber [12] with the following modifications: the stacking gel had 3.5% (w/v) acrylamide (38:1 acrylamide/ bis-acrylamide). The resolving gel had an acrylamide concentration gradient from 4.5 to 11.5 % (w/v) stabilised by a glycerol gradient from 8% to 16% (w/v). 12 mM Tris and 48 mM Glycine pH 8.5 were also included in the gel. Thylakoids at Chl concentration of 1 mg/ml were solubilised with 0.6 or 1 % of respectively α and β-DM. 30 µg of chlorophylls were loaded per each lane.
Purification of Photosystem I particles and LHCI
Photosystem I particles were purified from thylakoids by sucrose gradient ultracentrifugation upon solubilisation of thylakoid membranes with 1% β-DM, as described in [13]. To separate Lhc monomers, dimers and trimers Lhc fractions were concentrated, reloaded on a 0.1-1 M sucrose gradient and centrifuged in a SW60 Beckman rotor for 16 hours at 55000 rpm. LHCI and PSI core moieties were purified from PSI as described in [14].
Spectroscopy and pigment analysis.
The absorption spectra were recorded using a SLM-Aminco DK2000 spectrophotometer, in 5 mM Tricine pH 7.8, 0.2 M sucrose and 0.03% β-DM. HPLC analysis was as in [15]. Chlorophyll to carotenoid ratio and Chl a/b ratio was independently measured by fitting the spectrum of acetone extracts with the spectra of individual purified pigments [16].
SDS PAGE Electrophoresis and western blotting analysis
SDS-PAGE electrophoresis was performed as in [17], but using a acrylamide/bis-acrylamide ratio of 75:1 and a total concentration of acrylamide + bis-acrylamide of 4.5 % and 15.5 % respectively for the stacking and running gel. 6 M urea was also incorporated into the running gel. These modifications are optimised for the separation of Lhca1-4 from Arabidopsis thaliana. For gel staining, 0.05 % Coomassie R in 25% isopropanol, 10% acetic acid was used, in order to improve linearity with protein amount [18]. In the case of second dimension of non denaturing Deriphat PAGE, before loading gel slices were equilibrated for 30’ in 7M Urea, 2% SDS and 100 mM Tris HCl pH 6.8.
Stoichiometry evaluations based on Coomassie stain quantification
The protein amount was evaluated after SDS-PAGE by determining the amount of stain bound to each band by colorimetry. The gel image was acquired by using a Biorad GS710 scanner. The
picture was then analysed with Gel-Pro Analyzer© software, which quantifies the staining of the bands as IOD (Optical density integrated on the area of the band).
Reconstitution in vitro of Lhca1-4 monomeric complexes.
cDNAs of Lhca1-4 from Arabidopsis thaliana [9; 19] were expressed and isolated from the SG13009 strain of E. coli following a protocol previously described [20; 21]. Reconstitution in vitro were performed as described in [9].
Results
Arabidopsis plants depleted in individual Lhca1-5 proteins have been obtained from mutant collections or with an antisense approach. In particular, plants lacking Lhca1, Lhca4 and Lhca5 (respectively ∆a1, ∆a4 and ∆a5) are T-DNA insertion lines, while plants depleted in Lhca2 and Lhca3 (∆a2 and ∆a3) have been obtained with an antisense RNA inhibition [10; 22].
Non denaturing gels
In order to analyse the effect of the depletion of Lhca polypeptides we compared the composition of thylakoids membranes by separating pigment binding complexes into a non denaturing gel (Deriphat-PAGE) [12]. The result of the electrophoresis of thylakoids solubilised in very mild conditions (0.6 % α-DM) is shown in figure 1. In the picture the identification of each band in WT lane, based on their molecular weight, is also reported.
Figure 1. Deriphat PAGE profile of thylakoids from WT and
∆a1-a5 plants.
Thylakoids have been solubilised with 0.6 % α-DM before loading.
Bands detected in WT have been identified from their mobility and they are indicated on the left.
30 µg of Chls have been loaded in each lane.
From the picture, it can be seen that the whole profile in Lhca deficient plants is very similar to the WT, with the exception of the region involving PSI-LHCI complex. Thus, the lack of Lhca polypeptides has no detectable effects on Photosystem II subunits, as it was expected. The region of the gel where PSI-LHCI migrated is shown in more detail in figure 2A. In WT plants, one major PSI-LHCI band is present, while in Lhca deficient plants up to three bands can be resolved. In particular, we could recognise three classes of bands, based on their molecular weight. The slowest migrating band corresponds to the PSI-LHCI in WT, while the fastest corresponds to PSI core. The band with intermediate mobility (indicated as PSI-LHCI*), is composed by a Photosystem I with a reduced antenna complement. The identification of the different populations, reported in figure 2, was obtained by running a second dimension on SDS PAGE in denaturing conditions, which separates the component polypeptides from each green bands (as example see below figure 3).
Interestingly, the presence of a band corresponding to PSI core is detected in all ∆a1-a4 plants, but not in WT. This finding suggests that, when an individual Lhca polypeptide is lacking, a perturbation is induced in PSI-LHCI supercomplexes that leads to the complete loss of the LHCI moiety in at least a fraction of PSI particles. In ∆a4 plants PSI core is the most represented band, suggesting a wide destabilisation of LHCI in these plants. In ∆a1 plants, instead, the main band corresponds to a PSI-LHCI WT, while in ∆a2 and ∆a3 plants a band of PSI-LHCI* is the most evident. ∆a5 plants are very similar to WT and no major differences could be detected.
In the figure 2A, some other fainter bands can also be recognised in the different samples.
However, we could not identify them unequivocally because, using these very mild solubilisation Figure 2. Deriphat PAGE profile of thylakoids purified from WT and ∆a1-a5 plants, particular of Photosystem I region. Thylakoids have been solubilised with (A) 0.6% α-DM or (B) 1% β-DM before loading. Three different PSI bands have been identified by a second denaturing electrophoresis to be respectively PSI with full antenna (PSI-LHCI), PSI with reduced antenna (PSI-LHCI*) and PSI without any antenna polypeptide (PSI-core).
conditions, PSII supercomplexes are partially retained and co-migrate with PSI particles. As example, the bands migrating slightly faster or slower than the PSI-LHCI band in the lane loaded with WT membranes have been recognised as PSII particles by 2D analysis (not shown).
In order to have a clearer picture of the mutants pigment-protein pattern, we run a Deriphat PAGE after solubilisation of thylakoids membranes with 1% β-DM; the region involving PSI-LHCI is shown in figure 2B. This is a slightly harsher treatment, which dissociate completely PSII supercomplexes, but do not affect PSI-LHCI WT [13]. In WT lane, in fact, within this molecular weight range there is only one main band of PSI-LHCI, and only traces amount of PSI core are present. Therefore, we can be sure that, also in the case of mutants, all bands shown here derive from PSI. By using this stronger detergent treatment, we recognised bands belonging to the same three classes of mobility mentioned above, but their relative intensities changed with the stronger solubilisation conditions.
In ∆a1 plants all the three bands are visible: one has the same mobility as the WT PSI-LHCI, one of PSI core and one with intermediate mobility containing PSI with reduced antenna content (PSI-LHCI*). The first is due to a fraction of PSI-LHCI WT still present, due to the incomplete phenotype of the mutant that retains a residual level of Lhca1 [10]. In ∆a2 and ∆a3 plants, instead, only bands corresponding to PSI core and PSI with reduced antenna complement are visible. It is interesting to note that the PSI-LHCI* here is clearly less intense with β-DM solubilisation than with α-DM. Since the latter treatment is milder than the first, this finding suggests that PSI-LHCI*
is susceptible to detergent treatment and that with β-DM a larger part of the supercomplex dissociates into PSI core and free Lhca components. In ∆a4 plants, the main band is again the PSI core, as shown before also with α-DM solubilisation, but a weak band corresponding to PSI-LHCI*
is also visible. ∆a5 plants are again very similar to the WT, even if some faint bands with higher mobility can be recognised (PSI-LHCI* and PSI core), suggesting that the absence of Lhca5 is affecting a small fraction (at maximum 5%) of PSI particles.
Second SDS-PAGE dimension
As mentioned above, we run a second dimension on a denaturing SDS PAGE condition in order to analyse the polypeptide composition of bands from Deriphat PAGE. In figure 3, a particular of the second dimension of PSI region of a non denaturing gel with β-DM solubilisation is shown. From this gel, we could identify the polypeptides contained into the three classes of PSI bands described on Deriphat-PAGE. In the case of PSI-LHCI WT, the spots corresponding to Lhca1-4 are identified based on their mobility in this gel system [8]. In the same region of the gel, also a spot of a PSI core protein, PsaD, can be identified. The presence of this band is useful because it gives an indication of
the PSI core content. Bands of the non denaturing gel corresponding to PSI core can easily be identified because only this spot corresponding to PsaD is visible. As mentioned above, a band corresponding to PSI core is detected in all ∆a1-∆a4 plants. The most interesting information provided by this second dimension analysis regarded the PSI-LHCI* band. In fact, we could not only identify this band as a PSI with a reduced Lhca content, but also obtain information on the identity of Lhca polypeptides that are retained in this fraction from the different mutant lines. In fact, PSI-LHCI* from ∆a2 plants contained spots corresponding to Lhca1 and Lhca4, while spots of Lhca2 and Lhca3 were not detectable. The band from ∆a3 had the same composition suggesting that Lhca2 and Lhca3 cannot maintain their interaction with PSI core in the absence of each other.
Instead, in PSI-LHCI* from ∆a1, Lhca2 and Lhca3 are retained, while Lhca1 and Lhca4 are not detectable.
PSI-Lhca stoichiometry
In order to further characterise these mutants, PSI particles were purified with sucrose gradient centrifugation after 1% β-DM solubilisation. The sucrose gradient ultracentrifugation has a lower resolution power with respect to non denaturing gels: thus, the PSI with reduced antenna complement (LHCI*) evidenced from gels cannot be fully purified from PSI core (or PSI-LHCI in the case of ∆a1). The lowest part of PSI band was shown to be less contaminated and this was used for the following analyses. In figure 4A, the SDS PAGE profile of purified particles is shown. From a qualitative analysis of the picture, it could be driven that in ∆a1 plants the level of Lhca1 and Lhca4 is reduced with respect to PsaD, Lhca2 and Lhca3. In ∆a2 and ∆a3, instead, both
Figure 3. 2D SDS PAGE of PSI bands from WT and ∆a1-a5 plants (particular of region concerning Lhca polypeptides). The region involving PSI from Deriphat PAGE have been loaded in denaturing SDS PAGE. On the top the bands from native gel are reproduced and on the bottom the identification of bands from native gels is reported. PSI-LHCI particles from WT were also loaded on the left. The identification of polypeptides, based on their mobility, is reported.
Lhca2 and Lhca3 could not be detected, with the Coomassie staining. In ∆a4, instead, only small amounts of Lhca2 and Lhca3 are detectable, consistently with the presence of mostly PSI core in the non-denaturing gels. ∆a5 plants have a profile indistinguishable from WT consistent with the results from green-native gels. The same particles were analysed also by western blotting with antibodies against Lhca1-4 (not shown). By using this different detection method, some differences emerged: in fact, in ∆a2-PSI a residual fraction of Lhca3 was detected and in ∆a3 there was a small amount of Lhca2 left. These results are consistent with a parallel work done on PSI particles purified from the same plants (Klimmek et al submitted). The apparent discrepancy with figure 4A where the same particles are analysed with Coomassie staining is due to the different method of detection: the antibody is more sensitive than the Coomassie staining and it reveals the presence of a traces amount of polypeptides in the particles.
From the Coomassie stained SDS PAGE, we also evaluated quantitatively the stoichiometry of each Lhca by comparing the polypeptide content of mutants with the WT. This was done by measuring the amount of Coomassie blue bound to each band by densitometry, with a method described in [8].
The amount of each Lhca band was then related with the amount of PsaD and PsaF, which were used as internal standards for the quantification of PSI core amount. Knowing that in WT one copy of each Lhca1-4 per PSI core is present, we calculated the amount of Lhca polypeptides per PSI core present in each preparation, [4; 8]. Values resulting from these measurements obtained by this method are reported in figure 4B.
Figure 4. SDS PAGE analysis of PSI particles purified from WT and
∆a1-∆a5 plants. (A) Particular of SDS PAGE where Lhca polypeptides migrate. (B) Quantitative evaluation of Lhca1-4 polypeptides per PSI core in all samples. The stoichiometry was obtained by evaluation of Coomassie bound to each Lhca and normalisation to PsaD and PsaF content.
These data confirms the qualitative analysis exposed above, but also gives further interesting information. In fact, in ∆a1 plants, in addition the reduction of Lhca1 and Lhca4 already mentioned, there is a reduction of about the 40% of Lhca2 and Lhca3. In ∆a2 and ∆a3 plants, Lhca2 and Lhca3 are under the Coomassie detection limit and only about 40 % of Lhca1 and Lhca4 is retained. In
∆a4 around 10% of Lhca2 and Lhca3 are retained. In ∆a5 plants, instead, PSI is indistinguishable from WT within the experimental error. This quantification is not fully representative of the PSI particles in thylakoids membranes for two reasons: first, PSI-LHCI* is susceptible to detergent treatment and thus a non well-quantifiable fraction of Lhca are lost upon solubilisation. Second, sucrose gradient centrifugation is not able to fully exclude contamination of PSI-LHCI*. This inability can be remarked in the case of ∆a1 plants, where the residual content of Lhca1 in PSI particles is about 35% of WT, while in thylakoids the residual Lhca1 was estimated to be around 10%. In ∆a1 plants, in fact, PSI particles are enriched in the PSI-LHCI and then Lhca1 content is higher than in thylakoids.
This stoichiometric evaluation, however, is very interesting because it shows which polypeptides are affected by the absence of each polypeptide. Moreover, it shows that we always found Lhca1-4 and Lhca2-3 in similar amounts in all different samples. This strongly suggests the presence of a reciprocal dependence of one member of these two couples from the companion for the association to PSI core. Therefore, when one polypeptide is absent, the other one tend to be lost as well and it is not anymore bound to PSI core, at least stably enough to be co-purified in significant amounts.
LHCI stability is reduced in plants depleted in Lhca1-4 polypeptides
We noted already that the PSI-LHCI* population in ∆a2 and ∆a3 plants is susceptible to detergent treatment and that under stronger solubilisation conditions it is reduced, while the amplitude of the PSI core band is increased. Since the thylakoids used as starting material were the same, this effect must correspond to a release of Lhca from PSI upon β-DM solubilisation. These dissociated proteins should migrate in the upper part of the sucrose gradients, together with the antenna complexes of PSII. In order to verify this hypothesis, we analyse these fractions with western blotting using specific antibodies against Lhca1-4 polypeptides (figure 5A). The presence of Lhca2 and 3 is detected in both ∆a1 and ∆a4 plants, suggesting that in these plants a fraction of these polypeptides is detached from PSI core upon solubilisation. Symmetrically,Lhca1 and 4 are found in the upper band from both ∆a2 and ∆a3 plants. It is worth noting that in WT plants instead, Lhca are stably retained bound to PSI in these solubilisation conditions. Quite surprisingly, instead, ∆a5 plants showed some release of Lhca2 and Lhca4 polypeptides.
It is also interesting to determine if Lhca polypeptides are released from PSI as monomers or dimers. To this aim, we collected the free Lhc fraction (see figure 5B) and run it into an additional sucrose gradient for a longer time in order to increase resolution: as example, the result in the case of ∆a4 plants is also shown in figure 5C. A band with an intermediate mobility between monomeric and trimeric Lhcb was detected. This fraction contained dimeric Lhca complexes as detected by the presence of a red absorption tail due to the typical >700 nm spectral forms in the absorption spectrum (not shown), a spectroscopic fingerprinting of PSI antenna complexes. This finding confirms that a secondary loss of LHCI polypeptides is induced when one Lhca is missing and also suggests that these Lhca polypeptides are lost as dimers, thus confirming their capacity of interacting with each other within Lhca2/3 and Lhca1/4 couples.
Figure 5. Analysis of Lhca polypeptides released upon thylakoids solubilisation with 1% β-DM. A) Western Blotting analysis of free Lhc complexes fraction after solubilisation of thylakoids membranes. B) Sucrose gradient ultracentrifugation of ∆a2 thylakoid membranes solubilised with 1% β-DM. Five bands are indicated (B1-B5) and have been identified to be respectively: free pigments, monomeric Lhc, trimeric LHCII, PSII core, PSI-LHCI. Note that B4 and B5 are not well resolved. To avoid as much as possible contamination, the lowest part of the band was always used for analyses. The fraction between B2 and B3 (B2-3) is also indicated C) Second sucrose gradient of the B2-3 fraction. Bands corresponding to the different aggregation states are indicated.
Purification of Lhca1-4 dimer
LHCI polypeptides are very difficult to purify the one from the other, due to their biochemical similarity and to the presence of strong interactions between different polypeptides. Up to now, only a partial purification of Lhca1-4 dimer from Lhca2-3 could be achieved [9]. Therefore, most information available on individual properties of Lhca are derived mainly from reconstitutions in vitro [19; 23]. However, the recent structure of PSI-LHCI complex [4] recently showed the presence of “gap and “linker” chlorophylls bound at interfaces between PSI core and LHCI and between individual Lhca subunits. These binding sites are most probably not stable in monomeric Lhca reconstituted in vitro since protein-protein interactions are missing. For this reason, mutants lacking individual Lhca polypeptides are very useful tools for the characterisation of individual Lhca polypeptides because they could provide information on the extra pigments. A detailed spectroscopic analysis of PSI particles purified from these plants was reported in (Klimmek et al submitted). Here, we isolated LHCI from PSI particles: as expected from the characterisation of PSI-LHCI complexes, SDS PAGE of LHCI isolated from ∆a1 plants was enriched in Lhca2 and Lhca3. Opposite, in LHCI from ∆a2 and ∆a3 plants Lhca2 and Lhca3 polypeptides were not detected and then it contained almost pure Lhca1-4. From ∆a4 plants no LHCI fraction was obtained, since Lhca polypeptides retained in this mutant are too small. Lastly, from ∆a5 plants we obtained a LHCI fraction with a composition indistinguishable from the WT. The LHCI population isolated from all mutant plants (but ∆a4) migrated as dimers in a sucrose density gradient, as it was in the case of the WT. Therefore, this isolation yielded a LHCI preparation with WT composition (WT and ∆a5), one enriched in Lhca2-3 dimers (∆a1), and one containing Lhca1-4 dimers (∆a2 and
∆a3). It is then very interesting to analyse their spectroscopic and biochemical properties. In figure 6, absorption spectra of LHCI from ∆a1, ∆a2 and ∆3 plants are compared with WT. As it can be easily derived from the picture, spectra are all very similar. The only significant difference is detected in the regions corresponding to absorption of Chl b (around 470 and 650 nm). Here,
LHCI-∆a3 showed a slightly larger signal than in WT, while in LHCI-∆a1 it is smaller. This difference is confirmed by the analysis of pigment binding properties of LHCI populations: LHCI from ∆a1 plants has a slightly higher Chl a/b ratio (4.4 vs. 3.5 of WT).
The purification of LHCI also yielded PSI core preparations from all mutants. As expected, these preparations did not showed any difference depending on the genotype. However, it is interesting to compare the pigment binding properties of PSI core with the one of PSI particles purified from ∆a4 plants. Interestingly, the main difference is in the carotenoid content: in fact, PSI from ∆a4 plants showed the presence of significant amounts of violaxanthin and lutein, while in PSI core
preparation obtained by dissociating PSI-LHCI complex from WT, only β-carotene was found.
Recently, it was found that violaxanthin and lutein molecules are bound at interfaces between PSI core and antenna, in addition to gap and linker chlorophylls [8]. Therefore, these xanthophylls found in PSI of ∆a4 plants could be tentatively identified as a fraction of “gap carotenoids” that remains bound even in the absence of Lhc polypeptides.
Discussion
LHCI is co-operatively bound to PSI core
In this work, mutants of Arabidopsis thaliana depleted in individual Lhca complexes were biochemically characterised. From this analysis it emerged that in all ∆a1-∆a4 plants the binding of LHCI to PSI core was severely affected. In fact, with non denaturing electrophoresis, we always detected important amounts of PSI core without any associated Lhca, even in solubilisation conditions where the PSI-LHCI from WT is fully stable. Therefore, we can conclude that when one Lhca polypeptide is missing, the association of the remaining polypeptides to PSI-core is destabilised. We could still show the presence of PSI with associated antenna polypeptides (PSI-LHCI*), but this was only a fraction of the whole population. This residual population with an associated antenna is also susceptible to detergent treatment: this is evident if we compare the ∆a2 and ∆a3 profiles with α and β-DM solubilisation, the first one being milder than the latter. With α-DM, we detected a relevant amount of PSI with reduced antenna that is strongly reduced using β-DM. In addition, with a western blotting analysis, we found evidences in ∆a1-∆a4 plants, that Lhca polypeptides are detached upon solubilisation. We therefore conclude that, in the mutants, some antenna polypeptides are still associated to the PSI core within the thylakoid membranes but they are easily lost upon solubilisation.
This behaviour of mutant PSI-LHCI suggests the presence of strong protein-protein interactions between all Lhca proteins. When one subunit is missing, the binding of all the others to PSI core is weaker. The association of Lhca polypeptides to PSI core therefore appears to be highly cooperative.
Among this network of interactions stabilising the LHCI, Lhca4 appears to play a fundamental role for the interaction with the PSI core. In fact, when this polypeptide is missing, a very few Lhca polypeptides are found associated with PSI core. This effect could be due to its position in the middle of LHCI half moon [4] or to the presence of a critical interaction between Lhca4 and PsaF, the closest subunit of PSI core.
Lhca polypeptides also appear to behave like “couples”, consistently with their isolation as dimers [9]: in fact, Lhca1-4 and Lhca2-3 are not stably bound to PSI in the absence of their own “partner”.